The long term goal of this project is to understand the roles of DNA, proteins, RNA and nucleotides in the important process of mammalian transcription initiation. The specific aims for this project period include: 1. To identify the important events involving RNA polymerase II after it is recruited to mammalian promoters. To characterize the fundamental processes that drive polymerase to the stage where it can elongate transcript. 2. To identify and characterize the roles played by nucleotides, RNA, basal and activated factors, and ATPases in the opening of the promoter DNA, in the initiation of transcription, and in promoter clearance. 3. To explore the role of promoter diversity in regulating the properties of the promoter-bound RNA polymerases as they initiate, elongate and re- initiate transcription. 4. To establish procedures to accomplish these aims in experimental systems contain activation components. These aims will be accomplished by developing and applying assays for the processes that follow the recruitment of the polymerase to the promoter. They include permanganate assay for DNA melting, abortive initiation assay for initial RNA bond synthesis, Km and Ki profiles for the role of ATP- dependent processes, and biotinylated nucleic acid assays for promoter clearance events. These assays will be applied to several promoters that should represent a range of promoter-specific responses. The studies are necessary to approach the understanding and eventual treatment of the many health disorders that rack to defective components of the transcription apparatus.